Immunogenic recombinant yeast expression product and method for purifying it

ABSTRACT

Described is an immunogenic polypeptide that is a portion of the P. vivax circumsporozoite expressed by a recombinant yeast.

This patent application is a continuation-in-part application ofcopending U.S. patent application Ser. No. 754,645 filed on July 9, 1985in the names of Arnot, D. E. et al, and copending PCT application No.U.S. 86/01373 filed on June 24, 1986 both assigned to New YorkUniversity. Both of these applications are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates to an immunogenic polypeptide expressed by arecombinant yeast and comprising an amino acid sequence incorporating aportion of the P.vivax circumsporozoite (CS) protein including theregion of the repeat immunodominant epitope of said protein; and to amethod for purifying this polypeptide.

The immunogenic properties of the P.vivax CS protein and, in particular,those of a subsequence of this protein have been previously described,in the above-identified Arnot et al application and in Arnot, D. E. etal. Science 230:815-818, 1985 (also incorporated by reference).

In fact, the entire P.vivax CS gene has been identified and its sequencedescribed in the above-mentioned documents. As described therein, theP.vivax CS protein comprises a central region of 19 tandem repeats ofthe sequence:

    Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly

or, by another system of notation:

    D R A D/A G Q P A G

This region contains the repeat immunodominant epitope of the P.vivax CSprotein.

(The correspondence between the two notation systems is as follows:A=alanine, C=cysteine, D=aspartic acid, E=glutamic acid,F=phenylalanine, G=glycine, H=histidine, I=isoleucine, K=lysine,L=leucine, M=methionine, N=asparagine, P=proline, Q=glutamine,R=arginine, S=serine, T=threonine, V=valine, W=tryptophan, andY=tyrosine.)

Synthetic peptides consisting essentially of 18 amino acid residues(i.e. two repeats of the above repeating sequence and cyclicpermutations thereof--such as Asp-Gly-Gln-Pro-Ala-Gly-Asp-Arg-Ala) arerecognized by antibodies to the native P.vivax CS protein and in turngenerate antibodies (when injected in mammals) which recognize, and bindto, the native CS protein. Hence, such peptides have utility in avaccine against malaria.

One possible approach to making a vaccine against P.vivax malaria wouldbe to synthesize immunogenic peptides including a sequence correspondingto that of the immunodominant epitope of the P.vivax CS protein (i.e.,containing one and preferably two or more repeats). In the case ofP.vivax, however, because the sequence of the repeating unit of the CSprotein is rather long (in contrast to that of P.falciparum) classicalpeptide synthesis cannot be reliably used. Use of classical synthetictechniques to make longer peptides is especially inconvenient whenpracticed on a large scale, as would be necessary for manufacture of amalaria vaccine that would be distributed to millions of peopleworldwide.

Moreover, coupling of the synthesized peptide to a larger molecule thatwould play the role of a carrier or adjuvant would be necessary. Inaddition, in most instances, only a minor proportion of antibodies to asynthetic peptide recognize the same sequence in the native protein.That is, even if the synthetic peptide coupled to a carrier protein isshown to be immunogenic, most of the antibodies produced may not mediateprotective immunity against the pathogen. In this respect, the syntheticpeptide (NANP)₃ which is a candidate for preparing a vaccine againstP.falcioarum is exceptional, since at least 70% of the antipeptideantibodies recognize the malaria sporozoites. The explanation for thisunusual finding is probably that the (NANP)₃ peptide contains manyprolines (P) and asparagines (N), amino acids which are frequently foundin reverse turns of the protein molecule. Perhaps in this instance, apreferred configuration of (NANP)₃ in solution, mimics that of the samesequence in the native CS protein. However, from the examination of theamino acid sequence of the P.vivax repeats, it does not seem likely thata P.vivax peptide will behave similarly to (NANP)₃.

For these reasons, an alternative technique was sought for manufacturingan immunogenic peptide that could be used in a vaccine against P.vivaxmalaria. The present inventors looked to recombinant DNA and geneticengineering techniques to express immunogenic polypeptides that would beused to confer immunity to mammals against P.vivax malaria.

Although the technology was readily available for constructingrecombinant bacteria that would express a portion or all of therepeating amino acid sequence of the P.vivax protein, the presentinventors searched for an alternative expression system for thefollowing reasons:

First, the expression system should be reliable and able to produce thepolypeptide of interest consistently and with a high yield. Bacteria aanbe difficult to handle when produced in mass culture and overexpressinga foreign protein product.

Second, and more important, expression products of bacteria are oftendifficult to purify from pyrogenic impurities and other inflammatory andtoxic agents that either are co-expressed by the bacteria, or arenecessary additives in a bacterial growth medium.

Third, expression products of bacteria are most often fusion proteins,that is, they contain additional non-relevant sequences originating fromthe genes associated with the bacteria.

The present inventors looked to yeast expression systems, which havebeen substantially improved by recent advances in the field ofrecombinant DNA technology. Yeasts are hardier organisms than bacteriaand much easier to grow in mass culture. Moreover, recent advances inyeast genetics and cloning have increased the yields of yeast expressionsystems.

Purification of expression products of recombinant yeast systems is nota priori more complicated than purification of products of bacterialrecombinant systems and depends mostly on the characteristics of theparticular protein sought to be purified. Nevertheless, suchpurification is generally more complicated than that of a peptide orprotein produced by classical peptide synthetic techniques.

The present inventors chose a general yeast expression system that isthe subject matter of U.S. patent application Ser. No. 868,639 filed onMay 29, 1986 in the name of R. L. Burke et al and assigned to ChironCorporation. This application is incorporated by reference herein. Aportion of the P.vivax gene was chosen (for incorporation into the yeastorganisms) which included the entire tandemly repeated sequence plusanother segment preceding the repeat region (when the sequence is readfrom the N-terminal to the C-terminal). This segment incorporates asequence that is highly conserved in all malarial species and has beenpreviously found to be immunogenic in its own right. (See U.S. patentapplication Ser. No. 649,903 of V. Nussenzweig et al filed on Sept. 18,1984 assigned to New York University and incorporated by referenceherein.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide animmunogenic yeast-engineered polypeptide immunochemically reactive withmonoclonal antibodies against P.vivax circumsporozoite (CS) protein, anduseful in a vaccine preparation against malaria.

It is another object of the present invention to provide a method formaking the foregoing immunogenic polypeptide which could be practiced ona large scale and with a high yield.

Another object is to provide an immunogenic polypeptide suitable forincorporation in an anti-malaria vaccine preparation.

Another object is to provide an immunogenic polypeptide suitable for usein an anti-malaria vaccine preparation without being coupled to acarrier.

Another object is to provide a method for purifying the foregoingyeast-engineered polypeptide from an impure preparation thereofcomprising lysed yeast cell material and yeast culture media.

These and other objects of the invention will be apparent to thoseskilled in the art in view of the present specification, accompanyingclaims and appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the construction of the yeast expression vectorused in the present invention.

FIG. 2 is a map of yeast plasmid pAB24.

FIG. 3 is a polyacrylamide gel obtained using lysates from P.vivax/pAB24-transformed yeast and control yeast.

FIG. 4 is a Western analysis of lysates from yeast transformed with theP. vivax/pAB24 vector.

FIG. 5 is a plot of (a) the optical density profile of the materialpurified according to the invention (solid line); (b) the conductivityof the eluting buffer (solid line - black points); and (c) the percentactivity of eluted fractions in inhibiting the binding of antibody tonative CS protein.

FIG. 6 is a radioautograph of a SDS-PAGE gel demonstrating the purity ofthe engineered polypeptide when made and purified in accordance with thepresent invention.

FIG. 7 is a radioautograph of an isoelectric focusing gel showing theengineered polypeptide of the present invention.

FIG. 8 is a standard curve for a competitive radioimmunoassay and showsthe inhibition of binding of labeled, engineered CS polypeptide toanti-vivax CS protein antibodies by the presence of unlabeled engineeredpolypeptide of the present invention.

FIG. 9 is a plot of the amount of radiolabeled goat anti-mouse IgGrecognizing mouse antisera bound to engineered CS polypeptide, as afunction of the mouse serum dilution.

FIG. 10 is a plot of the binding of labeled mouse antisera (raisedagainst the engineered peptide) to immobilized synthetic 18-amino acid(repeat) vivax CS peptide, as a function of the antibody concentration.

FIG. 11 is a plot of the inhibition by synthetic vivax CS repeat peptideof the binding of mouse antisera to immobilized engineered CSpolypeptide prepared and purified according to the present invention, asa function of the concentration of the synthetic (inhibiting) peptide.

FIG. 12 is a plot of the binding of mouse antisera to immobilizedsynthetic non-repeat peptide, as a function of the antisera dilution.

SUMMARY OF THE INVENTION

This invention relates to a yeast-engineered polypeptide having an aminoacid sequence consisting essentially of the amino acid sequence:

    __________________________________________________________________________    A E P K N P R E N K L K Q P G D R A D G Q P A G D R A D                       G Q P A G D R A D G Q P A G D R A A G       Q                                                                             P                                                                             A                                                                             G D R A D  G                      Q P A G D R A D G Q P A G D R A D G Q       P                                                                             A                                                                             G                                                                             D R A D G  Q                      P A G D R A A G Q P A G D R A A G Q P       A                                                                             G                                                                             D                                                                             R A D G Q  P                      A G D R A A G Q P A G D R A D G Q P A       G                                                                             D                                                                             R                                                                             A A G Q P  A                      G D R A D G Q P A G D R A A G Q P A G       D                                                                             R                                                                             A                                                                             A G Q P A  G                      D R A A G Q P A G D R A A G Q P A G N       G                                                                             A                                                                             G                                                                             G Q A A G  G                      N A G G Q G Q N N E G A N A P N E K S       V                                                                             K                                                                             E                                                                             Y L D K V  R                      A T V G T E W T P                                                             __________________________________________________________________________

and produced by a method comprising the steps of:

(a) obtaining a 15 kilobase restriction endonuclease Bgl II P. vivax CSDNA fragment in a suitable vector;

(b) subcloning said fragment in a suitable vector;

(c) obtaining a 4.1 kilobase P. vivax DNA fragment coding for said aminoacid sequence;

(d) incorporating said fragment into a yeast expression vector,

(e) transforming yeast with said expression vector,

(f) culturing said yeast under conditions which allow expression of theprotein encoded by said vector, and

(g) harvesting the medium containing said expressed protein.

DETAILED DESCRIPTION OF THE INVENTION

The P.vavax CS gene is set forth below (together with the amino acidsequence for which it codes) ##STR1##

The DNA fragment chosen for insertion in the yeast host was: ##STR2##

This fragment includes the entire tandem repeat sequence plus a regionthat is substantially parallel to Region I of Dame, et al Science225:628 (1984), precedes the repeat region, and codes for the amino acidsequence A E P K N P R E N K L K Q P G.

This sequence includes the subsequence K L K Q P which is conserved inall malarial species that have been investigated to date.

The foregoing DNA fragment was obtained from the entire gene bysubcloning a 15 kb BglII fragment isolated as described by Arnot et al.(U.S. patent application Ser. No. 754,645, and Science 230:815-818, Nov.15, 1985) both incorporated by reference. It was then inserted in theDNA of modified yeast plasmid pAB24 as described in Example 1 below.

For expression of the P. vivax CS antigen in yeast, a hybrid promotercomprising the strong yeast glyceraldehyde-3phosphate dehydrogenase andthe glucose regulatable alcohol dehydrogenase-2 (ADH-2) promoter wasused. Fusion of this promoter to heterologous genes allows the growth ofyeast cultures to high density using glucose as a carbon source.Depletion of glucose in the media during fermentative growth leads toconcomitant induction of expression of the heterologous protein.Incorporation of the plasmid into high copy number, autonomouslyreplicating yeast plasmids, and transformation of yeast cells generatedstrains capable of expressing high levels of CS proteins on induction.

The yeast was grown in culture in YEP medium (1% w/v yeast extract, 2%peptone) with 1% glucose as described in Example 1 below. Two hundredliters of yeast material were thus obtained and stored at -80° C.

The thus obtained yeast material contained a complex mixture ofdifferent yeast proteins as well as culture medium additives. Gelelectrophoresis of this material on 7.5% sodium dodecyl sulfatepolyacrylamide gel gave an indication of its heterogeneity (see FIG. 6,lane 1). The expression procedure and plasmid construction are outlinedin FIG. 1 and described in Example 1, below.

It had been observed that all antibodies to the P. knowlesi CS proteinrecognized the immunodominant epitope region of this protein even afterthe CS protein had been heated at 100° C. for 30 minutes or subjected tocomplete denaturation by treatment with 6M guanidine and 1%beta-mercaptoethanol (Gysin, J., et al J. Exo. Med. 160:935, 1984;Godson, G. N., et al Nature 305:29, 1983).

This observation concerned the entire P. knowlesi CS protein and by nomeans established either that the P.vivax CS protein would show similarbehavior upon heating or that fragments thereof, consisting essentiallyof the polypeptide employed herein and encompassing the epitope region,would continue to be immunochemically reactive with anti-P.vivax CSprotein antibodies, after being subjected to heating conditions thatnormally result in denaturation of proteins. Another imponderable wasthat, in the present invention, the fragment of the CS protein was mixedwith very large amounts of non-relevant materials. Heating of themixture might lead to the formulation of aggregates with otherpolypeptides and masking of the epitopes and/or coprecipitation. It wasalso not known whether such fragments would continue to be immunogenicafter being subjected to the above heating treatment.

Nevertheless, the present inventors used a heating step as the initialstep in the purification of the yeast-engineered P.vivax CS polypeptide.(As used in this patent application, "polypeptide" will refer to arelatively long protein fragment, "protein" will refer to the entireprotein, and "peptide" will refer to a relatively short peptide, e.g.,one containing 30 amino acid residues or less. Regarding the use of theword "sequence", it will be understood that polypeptides and peptides inaccordance with the present invention will be functionally equivalent ifthey have the same sequence whether the sequence is set forth from the Nto the C terminus or from the C to the N terminus).

The mixture was then subjected to heating at 100° C., which resulted inmassive precipitation of lysed yeast cell material. The supernatant wasseparated, dried and lyophilized. The lyophilized supernatant residueshowed a substantial improvement in purity over the yeast extract (seeFIG. 6, lane 4).

A solution of the lyophilized material was then further purifiedsequentially by (a) anion-exchange chromatography using an electrolytegradient to elute the engineered CS polypeptide; and (b) molecular sievechromatography.

The fractions containing CS polypeptide activity were identified with aradioimmunoassay. A small amount of the highly purified yeast materialwas radiolabelled to a high specific activity with ¹²⁵ I. Each fractionwas assayed by a classical radioimmunoassay for its capacity to inhibitthe binding of the labelled material to immobilized anti-P.vivaxmonoclonal antibody, directed against the repetitive epitope of theP.vivax CS protein.

A major advantage of the purification process of the present inventionis its simplicity and its ready adaptability to scale-up. The thuspurified engineered P.vivax CS polypeptide is homogeneous by SDS-PAGEand isoelectric focusing and is thus expected to be substantially freeof pyrogenic, inflammatory and toxic impurities that may have beenassociated with the lysed yeast cell material and yeast culture media.

The yield of the combination of the yeast expression procedure and thepurification process of the present invention proved to be 13 mg of pureCS polypeptide per liter of yeast culture. Given that 200 liters of thisculture were produced in three days using pilot scale equipment (250liter fermenter), it is apparent that large amounts of the engineered CSpolypeptide can be made available in a short period of time.

A 200-liter stock of yeast extract contains sufficient engineered CSpolypeptide to immunize about 25,000 humans against P.vivax, using 100micrograms of polypeptide per person.

Major advantages of the CS polypeptide produced in accordance with thepresent invention include that the engineered peptide:

(a) can be produced in large scale free of impurities and ofnon-relevant antigens;

(b) represents a large fragment of the native CS molecule;

(c) has been shown to be highly immunogenic in rodents using aluminumhydroxide as an adjuvant;

(d) the antibodies which are produced react with both the "repeats" anda conserved region of the CS molecule.

Antibodies to the "repeats" have been shown to neutralize parasiteinjectivity very effectively. Antibodies to a peptide containing thisconserved region also neutralized parasite injectivity, but theinhibitory activity could not be accurately quantitated (Vergara, et al,J. Immunol. 134: 3445-3448, 1985). Moreover, the fact that the sequenceKLKQP, which is part of the peptide, is present in all CS proteins,suggests that this region has an important function.

The present invention is described in detail below by specific exampleswhich are intended to illustrate the invention without limiting itsscope.

EXAMPLE 1: Restriction of the P.vivax CS gene, Ligation into a Vector,Transformation of the Host Yeast Cells and Expression

In the following description all restriction endonucleases and enzymesare commercially available from Pharmacia Fine Chemical Co.,(Piscataway, N.J.), New England Biolabs (Beverley, Mass.), BoehringerMannheim (Indianapolis, Ind.) or Bethesda Research Laboratories, Inc.(Gaithersburg, Md.) and are used according to the manufacturer'sinstruction.

A pUC9 vector (Pharmacia Fine Chemical Co., Piscataway, NJ) containingthe P. vivax CS protein gene, was derived by subcloning a 15-kb BglIIfragment inserted into the BamHI sites of EMBL3, a bacteriophage lambdavector (as disclosed in Arnot et. al., U.S. patent application Ser. No.754,645 incorporated by reference). Clone pUC9Ci was digested with BglIIand XBaI and gel-purified to obtain a 4.1 kb fragment encoding all therepeat sequences (Arnot et. al., Science 230:815-818, 1985, incorporatedby reference.). This gel-purified fragment was then digested with FokIand BanI and ligated to the following 5'-phosphorylated syntheticlinkers synthesized by phosphoramidite method using AppliedBiosystems'380A DNA synthesizers. ##STR3##

Linker I provides for an NcoI site, while Linker II provides a SalIoverhang.

The linker-containing fragment was digested with NcoI and SalI, isolatedby gel electrophoresis and cloned onto NcoI/SalI digested pBS100(construction described below). The resulting plasmid is designatedpAG/P.vivaxl. pBS100 is a pBR322-derived plasmid containing the ADH-2regulated GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter(1200 bp) and GAPDH terminator (900 bp) (See FIG. 1) and itsconstruction is described below.

The ADH-2 portion of the promoter was constructed by cutting a plasmidcontaining the wild type ADH-2 gene (plasmid pADR2, see Beier and Young,Nature, 300:724-728 (1982) incorporated by reference with therestriction enzyme EcoR5, which cuts at a position +66 relative to theATG start codon, as well as in two other sites in pADR2, outside of theADH-2 region. The resulting mixture of a vector fragment and two smallerfragments was digested with Ba131 exonuclease to remove about 300 bp.Synthetic XhoI linkers having the sequence ##STR4## were chemicallysynthesized and ligated onto the Ba131 treated DNA. The resulting DNAlinker vector fragment (about 5 kb) was sparated from the linkers bycolumn chromatography (on Sepharose CL4B from Pharmacia), cut with therestriction enzyme XhoI, religated and used to transform E. coli toampicillin resistance. The positions of the XhoI linker additions weredetermined by DNA sequencing using standard techniques well known in theart. One plasmid which contained an XhoI linker located within the 5'nontranscribed region of the ADH-2 gene (position -232 from ATG) was cutwith the restriction enzyme XhoI, treated with single-strand specificnuclease S1, and subsequently treated with the restriction enzyme EcoRIto create a linear vector molecule having one blunt end at the site ofthe XhoI linker and an EcoRI end.

The GAP portion of the promoter was constructed by cutting plasmid pPGAP(as disclosed in European Patent Application No. 164,556 of ChironCorporation filed on May 3, 1985, incorporated by reference and accordedthe filing date of a corresponding U.S. patent application Ser. No.609,540 filed on May 11, 1984) with the enzymes BamHI and EcoRI,followed by the isolation of the 0.4Kbp DNA fragment. Plasmid pPGAP is ayeast plasmid containing a yeast GAPDH promoter and terminator sequenceswith flanking NcoI and SalI restriction endonuclease sites. The purifiedfragment was partially digested with the enzyme AluI to create a bluntend near the BamHI site and used to construct plasmid pJS104.

Plasmid pJS104 was constructed by the ligation of the AluI-EcoRI GAPpromoter fragment to the ADH-2 fragment present on the linear vectordescribed above.

The BamHI-NcoI ADH-GAP promoter fragment was obtained from plasmidpJS103, which is the same as pJS104 (supra) except that the GAP fragmentof the ADH-GAP promoter is about 200 bp in pJS103 and 400 bp in pJS104.Construction of pJS103 was the same as that for pJS104 except that the0.4 kb BamHI-EcoRI fragment was completely digested with AluI (insteadof partially digested for pJSI04) and a 200 bp fragment was isolated.

The entire above region containing the promoter, P. vivax segment andterminator, (hereinafter termed the "expression cassette") was excisedby digestion with BamHI, purified by gel electophoresis and cloned intoBamHI-digested pAB24. A restriction map of this plasmid is shown in FIG.2.

pAB24 is a yeast expression vector (FIG. 2) which contains the complete2 mu sequences necessary for an autonomous replication in yeast (Broach,in: Molecular Biology of the Yeast Saccharomyces, 1.445, Cold SpringHarbor Press, 1981) and pBR322 sequences. It also contains the yeastURA3 gene derived from plasmid YEp24 (Botstein, et al., Gene (1979) 8:17incorporated by reference) and the yeast LEU2^(d) gene derived fromplasmid pCl/1 (see European Patent Application Ser. No. 116,201 filed onAug. 22, 1984 in the name of Chiron Corporation, incorporated byreference). Insertion of the expression cassette was in the BamHI siteof pBR322, thus interrupting the gene for bacterial Resistance totetracyclin.

Expression of P. vivax CS proteins

Saccharomyces cerevisiae strain AB110 isolated by Chiron Corporation(Mat, leu2-04, or both leu2-3 and leu2-112, pep4-3, his4-580, cir.) wastransformed with pAB24/P.vivaxl-5 according to Hinnen, et al., Proc.Natl. Acad. Sci. USA 75:1929-1933 (1978). Single-transformant coloniesharboring GAP-regulated vectors were grown in 2ml of leu⁻(leucine-depleted) selective media to late log or stationary phase. Onlyyeast harboring the plasmid can grow in this medium. Cultures weresubsequently diluted 1:20 (v/v) in YEP (1% w/v yeast extract, 2% w/vpeptone) with 1% glucose, and grown to saturation (about 36h) in thismedium. Cells were lysed in the presence of sodium dodecyl sulfate (SDS)and dithiothreitol (DTT) and the lysates were clarified bycentrifugation. Cleared lysates were subjected to polyacrylamide gelelectrophoresis (Laemmli, U.K., Nature, 277:680, 1970). Followingstaining with Coomassie blue, a heavy band of about 38 kD was observedin extracts from transformants containing the P. vivax plasmid (FIG. 3).This band was detected in those cells transformed with the expressionvector, while being absent from extracts of cells harboring control(pCl/1) plasmids. The fusion protein accounts for over 10% of the totalcell protein. The reason for abnormal cell migration (38 kD versus 23kD, predicted from DNA construction) may be attributable to anomalousSDS binding as previously reported for P. Knowlesi CS proteins (Ozaki,et al., Cell 34:185, 1983), probably due to the low porportion ofhydrophobic residues.

To confirm the identity of the 38 kD band, proteins synthesized by yeastwere also submitted to Western analysis. Cleared yeast lysates preparedas described above were electrophoresed on polyacrylamide gels (Laemmli,supra) and proteins were subsequently electroblotted onto nitrocellulosefilters (Towbin, et. al., Proc. Natl. Acad. Sci. USA 76:3450, 1979). Thefilter was preincubated for 1h with 1% (BSA) in PBS and subsequentlytreated with a monoclonal antibody to P. vivax CS protein for 12h at 4°C. The filters were washed with 1% BSA/PBS and a second goat anti-mouseantibody conjugated with horseradish peroxidase (BioRad Laboratories,Richmond, Calif.) added. Finally, the filters were incubated withhorseradish peroxidase color development reagent (Bio-Rad, Richmond,Calif.) and washed. The Western analysis showed that the fusion proteinreacted with the monoclonal antibodies. (FIG. 4)

EXAMPLE 2 Purification of the circumsporozoite polypeptide of thePlasmodium vivax

The yeast cultures expressing a part of the circumsporozoite polypeptidewere prepared as in Example 1. The expressed polypeptide consisted of234 amino acids including all of the repeat domain and a conservedregion, namely Region I of Dame, J. B., et al, Science 225:628 (1984).The N-terminal amino acid of the expressed polypeptide is alanine in thenucleotide positions No. 385-7 (GCA) and the C-terminal amino acid isproline at nucleotide positions No. 1084-1086 (CCA); Arnot, et al,Science 230:815-818, 1985. The first step of purification of the peptidefragment expressed by yeast consisted of subjecting the extracts totemperatures of 100.C. The purification was performed as follows:

Extracts from pelleted yeast from 20 liters of yeast culture wereprepared in a bead beater, diluting the yeast in an equal volume of 0.1Msodium phosphate buffer, pH 7.3, 0.1% Triton-X, 1 mM EDTA, 1 mM PMSF(phenylmethylsulfonylfluoride) and 1 microgram per ml of pepstatin. Theextract (370 ml) was added to 200 ml of boiling water containing 1 mM ofPMSF and 1 microgram/ml of leupeptin. The mixture was brought to atemperature of 100° C. and kept for ten minutes with constant stirringat this temperature. The mixture was cooled to 0° C. and centrifuged at18,000 rotations per minute in a Beckman Ultracentrifuge Rotor TI-19,(Beckman Instruments, Palo Alto, Calif.) for fifteen minutes. Thesupernatant was removed and then lyophilized.

The dry material was dissolved in 120 ml of water, centrifuged to removea small residual amount of insoluble material, dialyzed extensivelyagainst distilled water for 48 hours and lyophilized again. The powderwas dissolved in 3 mM sodium potassium phosphate buffer, pH 7.5, and theconductivity adjusted with water to 0.58 mS. The solution wascentrifuged to remove insoluble materials and subjected to anionexchange chromatography in a DEAE-Sephacel (Pharmacia Fine Chem Co.,Piscataway, N.J.) column (5 cm×24 cm) equilibrated in the same buffer.The flow rate was adjusted to 100 ml per hour and 21 ml per tube werecollected. The column was then washed with 500 ml of the same buffer,i.e., with about one column volume. The elution continued with a bufferformed in a linear gradient in which 1,500 ml of of the initial bufferwere gradually mixed with 1500 ml of the same buffer also containing0.75M NaCl. The presence of the circumsporozoite polypeptide in thevarious fractions eluting from the column was detected using acompetitive radioimmunoassay described below.

The positive fractions eluted between fraction Nos. 6590 in asymmetrical peak with conductivities between 2 and 10 mS (FIG. 5). Thefull fractions 65-90 were lyophilized, redissolved in 60 ml of 0.3M NaCland dialyzed against 0.3M NaCl at room temperature for several hours.The dialyzate was centrifuged to remove a small amount of insolublematerial and one third, i.e. 20 ml, was subjected to molecular sievechromatography on Sephadex G-200 (Pharmacia) equilibrated with 0.3Msodium chloride. The Sephadex was superfine and the column was 5 cm indiameter and 100 cm long. Samples of 21 ml per tube were collected fromthe column. The CS polypeptide eluted in a sharp symmetrical peakbetween tubes 51-57. However, materials with higher and lower molecularweight in smaller amounts were distributed in tubes preceding andfollowing this peak. The contents of tubes 51-57 were pooled, dialyzedextensively against distilled water, and lyophilized. A total amount of89 mg of pure circumsporozoite polypeptides were recovered. On thisbasis, we calculated that the yield from 20 liters of yeast is 89×3,i.e., 267 mg of pure protein or about 13 mg per liter of yeast culture.The purity of the recovered circumsporozoite polypeptide was assessed bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andby isoelectric focusing under denaturing conditions. At concentrationsof 2 mg per ml, a single band with an isoelectric point of 4.3 wasdetected by isoelectric focusing (FIG. 7). By SDS-PAGE, a doublet wasdetected with a molecular weight between 43,000 and 45,000 (FIG. 6). Theextinction coefficient at 280 nanometers of solutions of the CSpolypeptide containing 1 mg of protein per ml was 0.2. A sample of thepolypeptide was subjected to partial N-terminal sequence analysis andthe major sequence was alanine-glutamicacid-proline-lysine-aspa-ragine-proline, as expected. Another sample wasradiolabelled with ¹²⁵ I and immunoprecipitated with monoclonalantibodies specific to the CS protein of Plasmodium vivax. Between 75and 85% of the counts were specifically recognized by the antibody.

A solid state competitive radioimmunoassay was used to detect theengineered CS polypeptide during the purification procedure. Thestandard curve relating the dose of antigen with the signal obtained inthe radioimmunoassay was prepared as follows. The purified, engineeredCS polypeptide was radiolabelled with ¹²⁵ I to a specific activity ofabout 2×10⁶ counts per minute per microgram protein using the well-knowniodogen method. Mixtures containing a constant amount of radiolabelledengineered CS polypeptide and variable amounts of purified cold (i.e.unlabeled) engineered CS polypeptide (in a total volume of 100microliters) were prepared. Thirty microliters of the mixtures were thendelivered to the bottom of wells of microtiter plates pre-coated withmonoclonal antibodies to the circumsporozoite protein of Plasmodiumvivax (2F2). This monoclonal antibody reacts with the repetitive epitopeof the circumsporozoite protein. (Nardin E. H. et al, J. Exo. Med.,156:20, 1982). The inhibitory effect of the cold peptide on the bindingof the labeled peptide to the bottom of the wells was proportional tothe concentration of cold peptide. This assay detected concentrations ofcold peptide as low as five nanograms per ml (FIG. 8). The assay of thecolumn fractions and of the extracts was performed exactly as describedabove and the degree of inhibition obtained was referred to the standardcurve (FIG. 8) to calculate the concentration of the CS protein peptide.All dilutions and washing in this assay were performed in a phosphatebuffer saline (PBS) containing 1.0% bovine serum albumin (BSA) and 0.1%sodium azide.

On the basis of the results of these assays, we calculated that theyeast cultures contain about 60 mg of circumsporozoite protein per literand that the recovery of the purified material was about 20% of thetotal. There were no losses in the initial step of purification, i.e.,following the boiling of the extracts this step removed more than 90% ofthe non-relevant protein bands observed in the SDS-PAGE of the originalextracts.

EXAMPLE 3: Immunization of mice with the yeast-engineered P.vivaxpolypeptide

The purified engineered CS polypeptide was then used as an antigen toimmunize mice. Ten outbred female Swiss-Webster, 8-12 week old mice wereinjected with 50 micrograms of the purified peptide adsorbed on aluminumhydroxide. Three and six weeks afterwards, the mice were boosted withthe same dose of antigen again using aluminum hydroxide as adjuvant. Tendays later, the mice were bled and the sera were subjected to analysisby an immunoradiometric assay to detect antibodies to the CSpolypeptide. In this assay, the wells of microtiter plates are coatedwith purified engineered CS polypeptide (10 micrograms/ml in PBS) andthen incubated the wells with 30 microliters of various dilutions of themouse serum in PBS-BSA. The wells were then washed and re-incubated with50,000 cpm of radiolabelled affinity-purified goat anti-mouseimmunoglobulin (10⁷ cpm/microgram of protein) diluted in PBS-BSA. Thewells were then washed again and counted.

All sera reacted with the engineered CS polypeptide at dilutions of1:10,000 or greater. The results of a titration of the pooled sera areshown in FIG. 9. That these sera contained large amounts of antibodiesto the "repeats" of the P. vivax CS protein was shown in two experimentsdescribed below.

(1) A synthetic 18-amino acid peptide (18-mer) comprising the sequence(Asp-Gly-Gln-Pro-Ala-Gly-Asp-Arg-Ala)₂ and representing two tandemrepeats of the P. vivax CS Protein was synthesized as described in (a)copending U.S. patent application Ser. No. 754,645 filed on July 9, 1985and incorporated by reference in the present application; and (b) Arnot,et al. Science, 230:815, 1985. This peptide was used to coat wells ofmicrotiter plates and the antibody contents of the sera detected by theimmunoradiometric assay described above. All sera reacted with titers at1:4,000 or above. The results of titrations of pooled sera obtainedafter the second antigen booster injection are shown in FIG. 10.

(2) The synthetic 18-amino acid peptide was also used to inhibit thebinding of antibodies, present in the pooled antiserum, to the wells ofplastic plates coated with the engineered CS polypeptide. In theseexperiments, we first incubated samples of a 1:3,000 dilution of thepooled serum from the mice (obtained after the 2nd booster rejection)with increasing concentrations of the 18-amino acid peptide describedabove. As a control, samples of serum were incubated with the sameconcentration of another 18-mer peptide with a different and unrelatedsequence of amino acids. Following an incubation of one hour at roomtemperature, 30 microliters of the mixtures were added to wells ofplates precoated with the engineered CS polypeptide. The wells were thenwashed, incubated with radiolabelled goat anti-mouse immunoglobulin, asdescribed above, washed again and counted. The results shown in FIG. 11demonstrated that the "repeat" peptide (but not the control peptide)inhibited about 50% of the reaction between the antibodies and the CSprotein. All dilutions and washings in these experiments were performedwith PBS-BSA.

The same sera were also analyzed for the possible presence of antibodiesto Region 1 of Dame, et al., suora. For this purpose, we used as animmobilized antigen a small synthetic peptide NH₂-Cys-Tyr-Asn-Glu-Lvs-Ile-Glu-Aro-Asn-Asn-Lvs-Leu-Lys-Gln-Pro-COOH. Thispeptide includes a sequence of five amino acids which are common to allCS proteins from all malaria parasites examined to date,Lys-Leu-Lys-Gln-Pro (Dame et al., Science, 225:593, 1984, and Enea, V.,personal communication). The first two amino acids (Cys and Tyr) areextraneous to the CS protein and were added for purposes of coupling thepeptide to a carrier protein and radiolabelling with ¹²⁵ I. This peptidewas used to coat wells of microtiter plates and the antibody contents inthe sera were detected as described above. After the second booster, allsera recognized this small peptide at dilutions of 1:2,000 or greater.The results of titrations of a pool of these sera is shown in FIG. 12.

The sera were also tested by indirect immunofluorescence using thesporozoite of P. vivax as the antigen. All sera were positive atdilutions of 1:1,000 or greater.

Finally, another experiment demonstrated that relatively low levels ofantibodies to the engineered CS protein neutralize the infectivity of P.vivax sporozoites. The assay was performed as described in J Immunol.132:909, 1984 (incorporated by reference) using the human hepatoma cellline Hep 62 as the target of parasite invasion. The results, set forthin Table I, below, show that a significant degree of inhibition wasobtained at serum dilutions of 1:5,000. The percentages of inhibitionswere calculated as 100-[(mean experimental values/mean ofcontrols)x100]. Controls consisted of sporozoites incubated withequivalent concentrations of normal (pre-immune) mouse serum.

                  TABLE I                                                         ______________________________________                                        INHIBITION OF P. VIVAX SPOROZOITES INTO HEPA-                                 TOCYTES IN VITRO BY ANTIBODIES TO THE YEAST-                                  ENGINEERED CS PROTEIN                                                         (Serum Dilution).sup.-1                                                                       % Inhibition                                                  ______________________________________                                          50            91                                                              250           91                                                            1,000           77                                                            5,000           42                                                            ______________________________________                                    

What we claim is:
 1. A polypeptide having an amino acid sequenceconsisting essentially of the amino acid sequence:

    ______________________________________                                        A E P K N P R E N K L K Q P G D R A D G Q P A G D R                           A D G Q P A G D R A D G Q P A G D R A A G Q P A G D                           R A D G Q P A G D R A D G Q P A G D R A D G Q P A G                           D R A D G Q P A G D R A A G Q P A G D R A A G Q P A                           G D R A D G Q P A G D R A A G Q P A G D R A D G Q P                           A G D R A A G Q P A G D R A D G Q P A G D R A A G Q                           P A G D R A A G Q P A G D R A A G Q P A G D R A A G                           Q P A G N G A G G Q A A G G N A G G G Q G Q N N E G                           A N A P N E K S V K E Y L D K V R A T V G T E W T                             ______________________________________                                    

and made by a method comprising the steps of: (a) obtaining a 15kilobase restriction endonuclease Bgl II P. vivax CS DNA fragment in asuitable vector; (b) subcloning said fragment in a suitable vector; (c)obtaining a 4.1 kilobase P. vivax DNA fragment coding for said aminoacid sequence; (d) incorporating said fragment into a yeast expressionvector, (e) transforming yeast with said expression vector, (f)culturing said yeast under conditions which allow expression of theprotein encoded by said vector, and (g) harvesting the medium containingsaid expressed protein.
 2. A polypeptide having an amino acid sequenceconsisting essentially of the sequence;

    __________________________________________________________________________    A E P K N P R E N K L K Q P G D R A D G Q P A G D R A D                       G Q P A G D R A D G Q P A G D R A A G       Q                                                                             P                                                                             A                                                                             G D R A D  G                      Q P A G D R A D G Q P A G D R A D G Q       P                                                                             A                                                                             G                                                                             D R A D G  Q                      P A G D R A A G Q P A G D R A A G Q P       A                                                                             G                                                                             D                                                                             R A D G Q  P                      A G D R A A G Q P A G D R A D G Q P A       G                                                                             D                                                                             R                                                                             A A G Q P  A                      G D R A D G Q P A G D R A A G Q P A G       D                                                                             R                                                                             A                                                                             A G Q P A  G                      D R A A G Q P A G D R A A G Q P A G N       G                                                                             A                                                                             G                                                                             G Q A A G  G                      N A G G G Q G Q N N E G A N A P N E K       S                                                                             V                                                                             K                                                                             E Y L D K  V                      R A T V G T E W T P.                                                          __________________________________________________________________________


3. The polypeptide of claim 2 expressed by a recombinant yeasttransformed by a yeast expression vector, said vector incorporating DNAcoding for said polypeptide.